Stem cell inhibiting proteins

Abstract

Proteinaceous molecules with stem cell inhibition activity are analogues of LD78 or MIP-1 alpha which have mutations to prevent or reduce multimer formation beyond certain stages (for example a dodecamer). Aggregate formation is therefore inhibited, and the resulting low molecular weight monomers (or oligomers) have improved solution properties leading to enhanced productivity and greater therapeutic utility as stem cell protective agents, which are useful in tumour therapy.

We claim: 
     
       1. A method of inhibiting proliferation of stem cells in a patient capable of benefitting from such inhibition, comprising adminstering to said patient a therapeutically effective amount of a composition comprising an LD78 or MIP-1α analogue of a wild-type LD78 or MIP-1α molecule, the analogue having stem cell inhibition (SCI) activity and being substantially incapable at physiological ionic strength of forming a stable multimer higher than a dodecamer as determined by Sedimentation Equilibrium Analytical Ultracentrifugation (AUC) and a pharmaceutically acceptable carrier. 
     
     
       2. The method of claim 1, wherein said analogue, which, relative to wild-type LD78 or MIP-1α, one or more amino acid residues involved in promoting and/or stabilizing association of the components of a dimeric, tetrameric, dodecameric or higher order complex is altered to have a lesser promoting and/or stabilizing effect. 
     
     
       3. The method of claim 2, wherein said alteration is a substitution. 
     
     
       4. The method of claim 1, wherein said analogue is substantially incapable at physiological ionic strength of forming a stable multimer higher than a tetramer. 
     
     
       5. The method of claim 4, wherein said analogue at physiological ionic strength forms a substantially homogeneous population of tetramers. 
     
     
       6. The method of claim 1, wherein said analogue is substantially incapable at physiological ionic strength of forming a stable multimer higher than a dimer. 
     
     
       7. The method of claim 6, wherein said analogue is substantially incapable at physiological ionic strength of forming a stable multimer higher than a monomer. 
     
     
       8. A method of inhibiting proliferation of stem cells in a patient, comprising administering to said patient a therapeutic effective amount of a proteinaceous molecule with stem cell inhibition (SCI) activity, the molecule being an analogue of a chemotactic cytokine superfamily molecule having SCI activity and a tendency to aggregate at physiological ionic strength, the analogue being substantially incapable at physiological ionic strength of forming a stable multimer higher than a dodecamer as determined by Sedimentation Equilibrium Analytical Ultracentrifugation (AUC), wherein the analogue is an LD78 analogue with a mutation at one or more of the amino acid residues with respect to wild-type LD78 selected from the group consisting of: Ser1, Leu2, Ala3, Ala4, Asp5, Thr6, Ala9, Phe12, Ser13, Tyr14, Ser16, Arg17, Gln18, Ile19, Pro20, Gln21, Phe23, Ile24, Asp26, Tyr 27, Phe28, Glu29, Ser31, Ser32, Gln33, Ser35, Lys36, Pro37, Gly38, Val39, Ile40, Leu42, Thr43, Lys44, Arg45, Ser46, Arg47, Gln48, Asp52, Glu55, Glu56, Gln59, Lys60, Tyr61, Val62, Asp64, Leu65, Glu66, Leu67, Ser68 and Ala69, and a pharmaceutically acceptable carrier. 
     
     
       9. The method of claim 8, wherein the mutation is a substitution and wherein there are only two mutation substitutions in said molecule. 
     
     
       10. A method of inhibiting proliferation of stem cells in a patient, comprising administering to said patient a therapeutic effective amount of a proteinaceous molecule with stem cell inhibition (SCI) activity, the molecule being an analogue of a chemotactic cytokine superfamily molecule having SCI activity and a tendency to aggregate at physiological ionic strength, the analogue being substantially incapable at physiological ionic strength of forming a stable multimer higher than a dodecamer as determined by Sedimentation Equilibrium Analytical Ultracentrifugation (AUC), wherein the analogue is an LD78 analogue and has at least one of the following substitutions with respect to wild-type LD78, wherein the substitutions are selected from the group consisting of: (a) Ile24>Asn, (b) Tyr27>Asn, (c) Phe28>Glu, (d) Glu29>Arg, (e) Lys44>Glu and (f) Arg45>Glu, and a pharmaceutically acceptable carrier. 
     
     
       11. The method of claim 10, wherein the analogue possesses both Lys44>Glu and Arg45>Gln substitutions. 
     
     
       12. A method of inhibiting proliferation of stem cells in a patient, comprising administering to said patient a therapeutic effective amount of a proteinaceous molecule with stem cell inhibition (SCI) activity, the molecule being an analogue of a chemotactic cytokine superfamily molecule having SCI activity and a tendency to aggregate at physiological ionic strength, the analogue being substantially incapable at physiological ionic strength of forming a stable multimer higher than a dodecamer as determined by Sedimentation Equilibrium Analytical Ultracentrifugation (AUC), wherein the analogue is an LD78 analogue and has at least one of the following substitutions with respect to wild-type LD78, wherein the substitutions are selected from the group consisting of: (a) Lys44>Glu with Arg45>Gln, (b) Arg47>Glu, (c) Phe28>Glu, (d) Phe28>Glu with Gln48>Glu, (e) Phe28>Glu with Arg47>Glu, (f) Arg17>Ser with Gln18>Glu, (g) Phe12>Ala, (h) Val39>Ala, (i) Ile40>Ala, (j) Asp26>Ala with Glu29>Arg and Arg47>Glu, (k) Arg17>Ser, (l) Glu29>Arg, (m) Gln18>Glu, (n) Asp26>Ser, (o) Gln48>Ser, (p) Thr15>Ala, (q) Gln21>Ser, (r) Phe23>Ala, (s) Ser32>Ala, (t) Ala51>Ser, (u) Ala4>Glu, (v) Phe12>Asp, (w) Asp26>Gln, (x) Lys36>Glu, (y) Lys44>Glu, (z) Arg45>Glu, (aa) Glu66>Gln, (bb) Phe12>Gln, (cc) Lys44>Ser, (dd) Arg17>Glu with Gln18>Glu, (ee) Asp26>Ala, (ff) Glu66>Ser, and, (gg) Ile19>Ala, and a pharmaceutically acceptable carrier. 
     
     
       13. The method of claim 12, wherein said molecule is an LD78 analogue having the following substitution with respect to wild-type LD78: Glu66>Ser. 
     
     
       14. The method of claim 12, wherein said molecule is an LD78 analogue having both the following substitution with respect to wild-type LD78: Ile19>Ala and Val39>Ala. 
     
     
       15. A method of inhibiting proliferation of stem cells in a patient undergoing chemotherapy and/or radiotherapy or in a patient suffering from hyperproliferative stem cell disorders, comprising administering to said patient a therapeutic effective amount of a proteinaceous molecule with stem cell inhibition (SCI) activity, the molecule being an analogue of a chemotactic cytokine superfamily molecule having SCI activity and a tendency to aggregate at physiological ionic strength, the analogue being substantially incapable at physiological ionic strength of forming a stable multimer higher than a dodecamer as determined by Sedimentation Equilibrium Analytical Ultracentrifugation (AUC), wherein the analogue is an LD78 analogue with a substitution mutation of aspartic acid to alanine at amino acid residues 26 with respect to wild-type LD78, and a pharmaceutically acceptable carrier. 
     
     
       16. A method of inhibiting proliferation of stem cells in a patient, comprising administering to said patient a therapeutic effective amount of a proteinaceous molecule with stem cell inhibition (SCI) activity, the molecule being an analogue of a chemotactic cytokine superfamily molecule having SCI activity and a tendency to aggregate at physiological ionic strength, the analogue being substantially incapable at physiological ionic strength of forming a stable multimer higher than a dodecamer as determined by Sedimentation Equilibrium Analytical Ultracentrifugation (AUC), wherein the analogue is an MIP-1α analogue with a mutation at one or more of the amino acid residues with respect to wild-type MIP-1 α selected from the group consisting of: Ala1, Pro2, Tyr3, Gly4, Ala5, Asp6, Thr7, Ala10, Phe13, Ser14, Tyr15, Ser16, Arg17, Lys 18, Ile19, Pro20, Arg21, Phe23, Ile24, Asp26, Phe28, Glu29, Ser3, Ser32, Leu33, Ser35, Gln36, Pro37, Gly38, Val39, Ile40, Leu42, Thr43, Lys44, Arg45, Asn46, Arg47, Gln48, Asp52, Glu55, Thr56, Gln59, Glu60, Tyr61, Ile62, Asp64, Leu65, Glu66, Leu67, Asn68 and Ala69, and a pharmaceutically acceptable carrier. 
     
     
       17. The method of claim 16, wherein the mutation is a substitution and wherein there are only two mutation substitutions in said molecule.